Figure 6.

14-3-3σ confers anoikis resistance via the activation of the ERK1/2 pathway. (A, B) The 14-3-3σ knock down HLF cells (A), and 14-3-3σ over-expressing HepG2 cells (B) were starved with serum-free medium for 24 h, then added EGF (40 ng/ml) and collected at the indicated time points. The levels of ERK1/2 and phosphorylated ERK1/2 were analyzed by western blot. Densitometry graphs of p-ERK/ERK of each cell line were shown. (C, D) The 14-3-3σ-knockdown and 14-3-3σ over-expressing cells were grown under suspension conditions. Cell lysates were prepared 48 h after suspension culture and immunoblotted with the indicated antibodies. (E) Western blot analysis of Bcl2, Bim, and GAPDH for HLF cells stably transfected with shNC or sh14-3-3σ, respectively, after detachment for 48 hours. (F) Western blot analysis of Bcl2, Bim, and GAPDH for HepG2 cells stably overexpressing 14-3-3σ after detachment for 48 hours. (G) HepG2 cells with 14-3-3σ overexpression were treated with ERK1/2 inhibitor U0126 (5 μM) and cultured in suspension for 48 h. The ERK and phospho (p)-ERK1/2 protein levels were then detected by western blot analysis. (H-L) HepG2-Vec and HepG2-14-3-3σ cells were treated with ERK1/2 inhibitor U0126 (5 μM) and cultured in the detached state for 48 hours. H and I, Cell apoptosis was assessed with flow cytometric analysis using Annexin V kit. J, Cell viability was assessed by Trypan blue exclusion assay. K, cell apoptosis was assessed by caspase 3 activity assay. L, The expressions of Caspase-3, cleaved-caspase-3, cleaved PARP, Bim, and Bcl-2 were assessed by western blot. (M-O) shNC, sh14-3-3σ, and 14-3-3σ-reconstituted HLF cells were cultured in suspension for 48 h and subjected to anoikis assay as above. M, western blot using indicated antibodies. N and O, flow cytometric analysis using Annexin V kit.